The development of novel pharmaceuticals faces a number of challenges, not least the problem of overcoming adverse toxicological effects. Indeed, adverse liver reactions remain the most prominent side effect. Metabolism and ultimate clearance of the majority of small molecule drugs occurs in the liver, and thus one of the main areas of focus in drug development concerns whether such compounds or their metabolites possess any hepatotoxic effect. Moreover, it is also of paramount importance to discover whether the secondary metabolites of such compounds also display any cytotoxic effects before the drug can begin clinical trial programmes.
Accordingly there is an urgent need for a model hepatic system that mimics human liver cells and that is able to predict effects of candidate molecules in the development of new drugs or chemicals. Traditionally, researchers have been forced to rely on primary liver-derived hepatocytes for such screening but these have a number of serious drawbacks including difficulty of maintaining the cells in long term culture and difficulty of obtaining consistent, homogeneous cell populations. A solution to this has been offered in the form of hepatocyte-like cells derived from human pluripotent stem cells. Human pluripotent stem cells (hPS) have already begun to revolutionise the ways in which relevant human cell types can be obtained. The possibility to indefinitely propagate pluripotent human embryonic-derived stem (hES) cells and human induced pluripotent stem (hiPS) cells and subsequently differentiate them into the desired target cell types is now providing a stable and virtually unlimited supply of cells for a range of applications in vivo and in vitro.
Unfortunately, currently available hepatocyte cell types do not always accurately model the hepatic environment, due to differences in morphology and function. For example, one often used alternative to primary cells are hepatic cell lines which often contain very low levels of (or totally lack) metabolising enzymes and have expression of other important proteins substantially different from the native hepatocyte in vivo. This is of particular relevance in relation to drug metabolism since one of the major deficiencies in hepatic cell lines is the absence or abnormally high expression of drug transporter proteins which are essential for drug screening purposes. Other available hepatic cell lines suffer from having a morphology and physiology which is more reminiscent of fetal or juvenile hepatocytes than the more clinically relevant adult hepatocytes. For these reasons there is a strong need to develop hepatocyte cell lines which are not only easy to culture and propagate but which also possess a more mature phenotype and which behave in a manner more akin to adult primary hepatocytes.
Derivation of hepatocyte-like cells from pluripotent stem cells is well established in the art. For in vitro purposes, several groups have developed protocols for deriving hepatocyte-like cells from hES cells (Hay et al., 2007; Hay et al., 2008; Brolen et al. 2010; Funakoshi et al. 2011) as well from hiPS cells (U.S. Pat. No. 8,148,151B; Song et al. 2009; Sullivan et al. 2010; Si-Tayeb et al. 2010; Chen et al. 2012). However, common to all of these is a specific low mRNA and protein expression of genes typical for mature hepatocytes, like phase I and II genes (e.g. CYP1A2, 2B6, 2C9, 2D6, 3A4), nuclear receptors (e.g. CAR and PXR), and other adult hepatic markers (e.g. Albumin). In addition, these hESC- and hiPSC-derived hepatocyte-like cells have high expression of fetal hepatic genes like α-fetoprotein (AFP) and CYP3A7, with the result that the cell types described therein have a fetal and not adult phenotype (for overview see e.g. Baxter et al. 2010). Furthermore, in most of the published studies on hESC- and hiPSC-derived hepatocyte-like cells, expression and functionality of drug transporters has not been investigated at all.
The modulation of RA signalling has been previously shown to be of importance during early hepatocyte differentiation and in particular at the stage when definitive endoderm (DE) is specified to become hepatic endoderm (Touboul et al 2010). Furthermore, RA-response elements have been identified in a number of genes important during early hepatocyte specification such as AFP and HNF4α (see Qian et al 2000; Magee et al 1998 and Hatzis et al 2001). However, at this early stage RA is known to have diverse effects and has also been found to be important in the derivation of pancreatic endoderm from pluripotent stem cells (Mfopou et al 2010). US Patent Application Publication US2012/0143316A1 discloses the use of all-trans retinoic acid in inducing hepatic differentiation from endoderm-like cells. As becomes evident, all of these disclosures relate to the modulation of RA signalling during endodermal and early hepatocyte differentiation. However, none of these documents teaches or suggests the applicability of retinoic acid as an hepatocyte maturation promoting agent, let alone its use at a late stage in hepatocyte differentiation.
The use of GSK 3 inhibitors have previously been described for early differentiation towards endoderm. WO08094597 (Dalton) describes a method of producing mesendoderm from primate pluripotent stem cells (pPSC) by contacting the pPSC with an effective amount of GSK3 inhibitor in a differentiation media. WO2007050043 (Stanton) describes a method for producing a mesodermal or an endodermal cell from a pluripotent stem cell, comprising a Wnt-signalling pathway in the pluripotent stem cell. US2006003446 (Keller) describes a way of making a cell population enriched for endoderm cells culturing embryonic stem cells in the absence of serum and in the presence of activin and an inhibitor of Wnt-signalling. Modulation of Wnt signalling through the use of a GSK3 inhibitor has also been shown to be beneficial in specifying hepatocyte cell fate when DE cells are exposed to this treatment (WO2011/116930). Again, all of these disclosures relate to modulation of GSK3 signalling at relatively early stages in endodermal or hepatic specification.
Culturing of cells on certain matrix components has been known to affect their growth and, in the case of multipotent cells, to affect their ultimate differentiation. For example, pluripotent stem cells have been shown to undergo epithelial to mesenchymal transition and thence develop into cardiac cell types through overlaying of the stem cells with certain matrix components (WO2011060342). Moreover, culturing of adult primary hepatocytes in defined “sandwich” of matrix components has long been known to help them maintain their phenotype and metabolic activity (Dunn et al 1991), (Page et al 2007).